There is currently a surge of activity in developing synthetic nonviral gene delivery systems for gene therapeutic applications because of their low toxicity, nonimmunogenicity, and ease of production. Cationic liposome-DNA (CL-DNA) complexes have shown gene expression in vivo in targeted organs, and human clinical protocols are ongoing. Moreover, the single largest advantage of nonviral over viral methods for gene delivery is the potential of transferring extremely large pieces of DNA into cells. This was clearly demonstrated when partial fractions of order Mega base pairs of human artificial chromosome (HAC) was transferred into cells using cationic liposomes (CLs) as a vector although extremely inefficiently. However, because the mechanism of action of CL-DNA complexes remains largely unknown, transfection efficiencies are at present very low and vary by up to a factor of 100 in different cell lines. The low transfection efficiencies with nonviral delivery methods are the result of poorly understood transfection-related mechanisms at the molecular and self-assembled levels, in particular, a general lack of knowledge of structures of CL-DNA complexes and their interactions with cellular components inside animal cells which lead to gene release and expression. The aims of this research application are (1) to explore the various self-assembled structures in CL-DNA complexes and to identify the critical parameters which control the intermolecular interactions and give rise to the structures, and (2) to determine the relation between the CL-DNA complex structures and transfection efficiency in animal cell culture. To achieve the goals of this application we will use state-of the-art techniques involving synchrotron x-ray scattering and diffraction (at the Stanford Synchrotron Radiation Laboratory) and video-enhanced optical microscopy to probe the structures of CL-DNA complexes and their interactions with animal cells. The structures will be correlated to transfection efficiencies by modern molecular biology methods of quantitatively measuring expression of the luciferase reporter gene in animal cells. The broad long-range goal of the research is to develop optimal synthetic nonviral carriers of DNA for gene therapy and disease control.